EPF and the mitochondrial chaperonin 10 have identical amino acid sequences (SEQ ID NO:4) although they may be encoded by different genes (Summers et al., 1998) and have very different physiological functions. Moreover, EPF is a secreted peptide, whereas chaperonin 10 is found in the intracellular vesicles along the secretory pathway.
Chaperonin 10 belongs to the family of heat shock proteins. In the mitochondria it forms a chaperonin complex with heat shock protein 60 which is important for mitochondrial protein folding and function. Upon ischemia, upregulation of these two proteins is able to protect brain tissue as well as cardiac myocytes (Lau et al., 1997) against ischemia/reperfusion injury (Hickey et al., 2000).
Up until now, EPF is mainly known as an important factor in embryonic development, in the pre-implementation stage as well as in the peri-implantation stage (Athanasas-Platsis et al., 2000). Its importance in these two phases is based on its growth regulatory and immunomodulatory properties. These actions of EPF are also apparent as it is produced by proliferating primary and neoplastic cells, where it functions as an autocrine growth factor both in vitro and in vivo (Morton, 1998).
The presence of EPF has been repeatedly confirmed as indispensable to successful pregnancy. Recently, Cheng S J et al. (Am. J. Reprod. Immunol. 2000 October; 44(4):211-3) demonstrated that the EPF level declined rapidly after surgical abortion and as such suggests that monitoring the EPF activity is a useful index for embryonic care and development of normal pregnancy. Accordingly suppression of EPF activity could have a contraceptive purpose, whilst enhancing EPF activity may prevent foetal loss.
EPF is already secreted into maternal serum within 6-12 hours after fertilisation, and EPF or EPF-derived or -related peptides could therefore be a useful early marker for diagnosing pregnancy. Such diagnostic would be useful in human medicine but also for veterinary applications. Currently used EPF assays (e.g. rosette test) are cumbersome and unreliable. Moreover, they do not distinguish between various forms of EPF. Tests based on antibodies specific for (poly)peptides with EPF activity or assays that distinguish the various forms of bioactive EPF (e.g. based on chromatography and/or mass spectroscopy) may therefore offer significant advantages. EPF activity has been repeatedly confirmed as indispensable to successful pregnancy. Recently, Cheng S J et al. (Am. J. Reprod. Immunol. 2000 October; 44(4):211-3) demonstrated that the EPF level declined rapidly after surgical abortion and as such suggests that monitoring the EPF activity is a useful index for embryonic well-being and development of normal pregnancy. Accordingly, suppression of EPF activity could have a contraceptive purpose, whilst enhancing physiological or correcting abnormal EPF activity may prevent foetal loss.
It has been observed that pregnancy has a positive influence on the development of multiple sclerosis by decreasing the rate of relapses during pregnancy (Confavreux et al., 1998). As pointed out by Morton (1998), EPF is ‘considered to be one of the major factors involved in the modification of multiple sclerosis observed during pregnancy’. The positive effect of the immunosuppressive action of EPF during pregnancy could also be observed for another auto-immune disease, rheumatoid arthritis (Davis and Maslow, 1992).
The potential activities of EPF and chaperonin 10 in human systems make the peptides a target for studies aimed at identifying compounds that enhance or diminish the biological effects of the peptides. However, while binding sites for EPF and chaperonin 10 were shown to exist, no receptor for these peptides was identified. The lack of a known receptor has hampered the design of assays and impeded efforts to discover and study compounds that mimic or alter the biological effects of EPF or chaperonin 10.
The present invention solves this problem in the art as reverse pharmacology has led to the identification of the human Dorsal Root Receptors (hDRRs) as receptor proteins for EPF or chaperonin 10. With this finding the present inventors further substantiate the immunomodulatory properties of EPF as hDRR receptors are shown to be expressed in lymph nodes and chromosomal mapping of the hDRRs to chromosome 11p15 linked them to a number of lymphoblastic leukemias. It further provides assays to identify compounds that mimic or alter the biological effects of EPF or chaperonin 10, as well as the pharmacological use thereof.
hDRRs belongs to the family of G protein coupled receptors (GPCRs) which share a common structural organization characterized by an extracellular N-terminal end, seven hydrophobic alpha helices putatively constituting transmembrane domains and an intracellular C-terminal domain. GPCRs bind a variety of ligands that trigger intracellular signals through the activation of transducing G proteins (Caron et al., Rec. Prog. Horm. Res. 48:277-290 (1993); Freedman et al., Rec. Prog. Horm. Res. 51:319-353 (1996)).
Recent reviews (Stadel et al., 1997; Wilson et al., 1998) count the number of GPCRs being used as a target for commercially useful drugs to 25, this constitutes 18% of the 140 characterized cloned human GPCRs. Extrapolation from fully sequenced genomes (yeast, C. elegans) to the expected 30.000 human genes leads to the expectation that 5000 human GPCRs will be found in the course of the next 3 years. In analogy to the current numbers, 150 of these novel orphan GPCRs should develop into a target of a commercially interesting drug during the next decade. This calculation does not take into account that additional characterized GPCRs are currently the basis of compounds in development.
In general, it is fair to say that reverse pharmacology on orphan GPCRs will deliver novel approaches for the treatment of various diseases, which will outcompete current strategies or enable treatment of conditions that cannot be treated by current means. This has been recognized by the pharmaceutical industry as is documented by recent publications on orphan GPCR ligand identification (orphanin FQ (Reinscheid et al, 1995.), orexin (Sakurai et al, 1998), prolactin-releasing peptide (Hinuma et al.), apelin (Tatemoto et al., 1998).
The nucleic acid and polypeptide sequences of the human Dorsal Root Receptors 1-6 were described in PCT application WO 99/32519 A1 published Jul. 1, 1999. Based on their homology to the rat Dorsal Root Receptor, this document describes the hDRR receptors as being involved in transmission, modulation and sensation of pain, including the use thereof in assays for the identification of new agents for anesthesia and analgesia. In this PCT application the Dorsal Root Ganglia localization has been confirmed for hDRR5 in fetal dorsal root ganglia. However, for none of the human adult tissues examined thus far, including dorsal root ganglia, a hDRR specific hybridization signal could be detected. In addition for none of the disclosed hDRR receptors in this application (WO 99/32519 A1) a natural ligand could be identified. The characterization of hDRR4 as an angiotensin receptor in aforesaid application (WO 99/32519-A1), based upon the stimulation of this receptor with angiotensin II and III, could not be confirmed by the present inventors.
For another human Dorsal Root Receptor, hDRR7, the nucleic acid and polypeptide sequences were described in PCT applications WO 01/16159-A1 published Mar. 8, 2001 and in WO 01/19983-A1 published Mar. 22, 2001. In neither of these documents a ligand for the hDRR7 receptor has been identified. In PCT application WO 01/16159-A1 the hDRR7 receptor is referred to as TheAnt disclosing a large number of conditions associated with said polypeptide. However, no substantive evidence is provided to link this receptor to any of the enlisted disease states. In PCT application WO 01/19983 the hDRR7 receptor is generally described as a GPCR with low sequence similarity to the somatostatin 3 receptor.
Therefore, no assay employing competition with a natural ligand for hDRRs, or employing a comparison to the interaction of the hDRRs with a natural ligand has been described. The latter is essential to use the hDRRs as pharmacological tools to explore receptor function and relationship to disease states. The present invention solves this problem in the art and provides assays that employ the interaction of hDRRs and EPF or EPF-related peptides, to determine whether a candidate compound is a ligand, agonist or antagonist of hDRRs.
Only recently, Lembo et al. (Nature Neuroscience 5, 201-209 (2002)) demonstrated that the hDRR 4 receptor, also known as Sensory Neuron—Specific G protein coupled—Receptor 4 (SNSR4) or Mas Related Gene Receptor 1 (hMrgX1), are potently activated by gene products of the opioid peptide precursor proenkephalin A. In particular BAM22 and BAM22 fragments, known to be involved in the control of nociception, were shown to activate the hDRR in a FLIPR based calcium assay.
In the present invention EPF and EPF-related peptides were shown to activate both the hDRR4 and hDRR7 receptors. Further, hDRR4 was shown to be predominantly expressed in dorsal root and trigeminal ganglia (Lembo et al., Nature Neuroscience 5, 201-209 (2002)), whilst for hDRR7 it has now been demonstrated that this receptor is predominantly expressed in lymph nodes. This receptor specific expression pattern suggests a different functionality in response to ligand activation.
It would accordingly be beneficial if one could design receptor-specific compounds useful in treating receptor related disorders. The identification of EPF and EPF-related peptides as ligands for hDRR4 and hDRR7, now provides a basis for the development of in vitro screening methods to identify compounds capable of modulating a hDRR involvement in the transmission, modulation and sensation of pain. Furthermore, they are useful as anticonceptives, in anesthesia and analgesia and in identifying compounds capable of modulating hDRR mediated disorders such as rheumatoid arthritis, multiple sclerosis or other conditions where immunosuppressive actions are desired like Inflammatory Bowel Disease (IBD) or to prevent transplant rejection.
These and other aspects of the invention are described herein in more detail.